Tutorial on chick early development

(reprinted from Stern,
C.D.
(2004). Gastrulation in the
chick. In: Gastrulation:
from cells to embryo. (ed. C.D. Stern). Cold Spring Harbor
Press. pp. 219-232. Copyright Claudio D Stern and Cold Spring Harbor
Press) - please cite this reference if using any
of this information. (if you want to reproduce figures
you need to contact both the author and the publishers)

Early
stages: blastoderm
formation and early polarity

Avian eggs and early embryos differ in several
respects
from the majority of vertebrates, yet many of the principles governing
the
early steps of development are very similar. Unlike amphibians and some
other
embryos, the point of sperm entry is not a crucial determinant of
polarity of
the early embryo, because avian embryos are highly polyspermic – as
many as
5-26 sperm may enter the egg in domestic fowl (chicken) while turkey
eggs may
be entered by several hundred sperm heads (Waddington et al., 1998;
Stepinska and Olszanska, 2003).
Cleavage, as in most species containing a large
amount of yolk (Arendt and
Nübler-Jung, 1999)
(Figs. 1-2), is meroblastic – that is, cleavage
occurs within a planar disc, and new cell membranes open into the yolk
generating small cells in the center and large, yolk-laden, open cells
at the
periphery (Fig. 2 B, C) (Bellairs et
al., 1978).
Unfortunately these
stages are difficult to study because cleavage occurs while the egg is
still
within the maternal oviduct and before the shell is deposited (Fig. 1)
– in the
chicken, laying occurs some 20 hours post-fertilization, when the
embryo is a flat
disc (blastodisc or blastoderm) containing at least 20,000 cells.

A
rule of
thumb (von Baer’s rule) can help to predict the orientation of the
head-tail
axis of the embryo from the outside of the egg: with the egg lying
along its
long axis, and its blunt end to the operator’s left, the axis of the
embryo
will run at right angles to the long egg axis with the head pointing
away from
the operator. However this rule only applies in about 60-70% of cases.

As
in teleosts, which are also meroblastic, maternal determinants are
likely to
exist also in avian embryos. One such determinant is called δ-ooplasm
(or
subgerminal ooplasm) – this is contained in the thin, “white” yolk that
makes
up the latebra and the nucleus of Pander (Fig. 2 A, C). The former is a
funnel-like
structure extending from just under the blastoderm to the center of the
yolk (Callebaut et al., 1998a; Callebaut et
al., 1999a; Callebaut et al., 2000a).
We do not know the molecular nature or the functions
of this ooplasm or whether it plays any role in polarity, although it
has been
suggested that it determines the position from which the endoblast and
Koller’s
sickle (see below) will form (Callebaut,
1993; Callebaut et al., 1998a; Callebaut et al., 2001).

Avian
embryos appear to generate bilateral symmetry under the influence of
gravity (Kochav and Eyal-Giladi, 1971; Callebaut,
1978; Eyal-Giladi and Fabian, 1980; Callebaut, 1993; Eyal-Giladi et
al., 1994;
Callebaut et al., 2001).
As the egg descends along the oviduct, it rotates
with the blastoderm remaining at an angle of about 45o to
the
vertical – the lower edge of the blastoderm will become the future head
end.
However, we are still completely ignorant about the mechanism by which
gravity
breaks radial symmetry. It was suggested that opposite (upper and
lower) poles
of the disc are exposed to gravitational forces of different magnitude
and that
this causes differential amounts of cell shedding (Kochav
and Eyal-Giladi, 1971; Eyal-Giladi and Kochav, 1976;
Eyal-Giladi and Fabian, 1980; Eyal-Giladi et al., 1994).
However, this has never been demonstrated and the
alternative hypothesis that rotation exposes the poles of the
blastoderm to
subgerminal ooplasm to different extents (Callebaut,
1993)
seems much more likely.

Importantly,
neither gravity nor maternal determinants irreversibly fixes bilateral
symmetry
until gastrulation starts, because avian embryos are highly regulative – right up to the time of appearance of the
primitive streak, blastoderms can be split into several pieces (pie
slices)
each of which can spontaneously generate a complete embryonic axis (Lutz,
1949; Spratt and Haas, 1960a; Callebaut and Van Nueten, 1995).
Therefore, gravity and localized maternal components
can, at best, only bias polarity but do not act as definitive
determinants.

The blastoderm stage

By the time the egg
is laid, the
embryo is an almost flat disc in which an inner area pellucida can be
distinguished from a more peripheral ring, the area opaca. Closest to
the
acellular vitelline membrane that envelops the yolk (“dorsal” side), a
simple,
one-cell-tick epithelium is continuous over both areas (Fig. 3) (Bancroft and Bellairs, 1974; Bellairs et
al., 1975).
This is the epiblast. At this stage the cells of the
epiblast of the two concentric areas are almost indistinguishable
morphologically except that at the very edge of the area opaca the
cells are
flattened and contact the vitelline membrane, against which they will
later
spread and help expand the blastoderm; the center of the disc is not
attached
to the membrane. At later stages however cells of the area opaca
epiblast
become less columnar than those of the area pellucida.

Deep
(facing the yolk) to the epiblast the cellular composition is more
complex. The
area opaca contains several layers of large (up to 150-200μm) yolky
cells;
those closest to the epiblast are firmly attached to it. This is the
germ wall.
By contrast, the center of the disc (area pellucida) does not yet
contain a
continuous cell layer, but is peppered with small islands of about 5-10
cells
each. These are also yolky but not as large as the deep part of the
area opaca
(about 100μm). The islands may arise by a process of polyingression (or
shedding) that occurs throughout the area pellucida shortly before
laying (Peter, 1938; Kochav et al., 1980; Fabian
and Eyal-Giladi, 1981; Eyal-Giladi, 1984),
but the fate of the shed cells has never been
studied experimentally. The islands will later fuse with each other to
generate
the primitive endodermal layer, or hypoblast (“entophyll” or “primary
hypoblast” in the earlier literature).

Between
the
area opaca and the area pellucida is a narrow region (known as the
marginal
zone). The epiblast of this region (to which the term refers) is not
distinguishable from other regions of epiblast, except for the
expression of Vg1
at its posterior end (posterior marginal zone; see below) (Seleiro et al., 1996; Shah et al., 1997)
and a slight gradient of cWnt8C decreasing
from posterior to anterior (Skromne
and Stern, 2001).
The only morphological landmark is that the deep
part (germ wall) is not strongly attached to the epiblast, unlike the
area
opaca. This region is known as the germ wall margin. In carefully
dissected
blastoderms it forms a lip that protrudes under the area pellucida for
a few
cell diameters (Stern and Ireland,
1981; Stern, 1990).

The
boundary between area pellucida and marginal zone is marked, at the
future
posterior edge, by a crescent-shaped ridge of small cells, tighly
adherent to
the epiblast – Koller’s sickle (also known as Rauber’s sickle) (Koller,
1882; Callebaut and Van Nueten, 1994),
which expresses goosecoid (Izpisua-Belmonte et
al., 1993).
Together, these components define a blastoderm of
stage X (Roman numerals from I-XIV are used to classify stages before
formation
of the primitive streak according to Eyal-Giladi
and Kochav, 1976;
Arabic numerals from 2 onwards are used for
post-streak embryos following Hamburger
and Hamilton, 1951).

In
the following few hours of incubation the islands of hypoblast
gradually fuse
together, probably by a process of flattening of the cells, which
proceeds from
posterior to anterior to generate a continuous but relatively loose
layer, the
hypoblast proper (Vakaet, 1970;
Stern, 1990)
(see supplementary movie {movie1} and
animation
{movie2}). This
layer covers half of the area pellucida at
stage XII and almost all of it at stage XIII (Fig. 3). Shortly after,
two
changes take place: first, the posterior germ wall margin cells and
their
progeny start to move centripetally (Stern,
1990)
and displace the hypoblast anteriorly; this new layer
is the endoblast (or “sickle endoblast” or “secondary hypoblast” in the
earlier
literature). Hypoblast and endoblast can be distinguished by several
markers
including goosecoid (in White Leghorns and some other strains),
Hex,
Hesx1/Rpx, Cerberus/Caronte, Otx2 and Crescent, all of
which are
expressed in the hypoblast but not in the endoblast (Bachvarova
et al., 1998; Foley et al., 2000; Bertocchini and
Stern, 2002).
The hypoblast is therefore similar to the anterior
visceral endoderm (AVE) of the mouse embryo. At the same time, a
posterior thickening (the posterior bridge),
apparently derived from Koller’s sickle appears – this transient
structure
defines stage XIV, and the primitive streak starts to form immediately
thereafter. None of the components of the deep layer (hypoblast,
endoblast,
germ wall or its margin) contribute to any embryonic tissues – they
only
generate extraembryonic membranes such as the yolk sac stalk, and later
disappear.

Fate maps and cell movements at the blastoderm stage

Many authors have
constructed
fate and specification maps of the epiblast of the chick at the
blastoderm
stage (Rudnick, 1935, 1938; Hatada
and Stern, 1994; Callebaut et al., 1996; Bachvarova et al., 1998).
The most detailed ones (Hatada and Stern, 1994)
reveal an orderly arrangement of prospective
embryonic tissues which gradually changes with time (Fig. 4), due to
extensive
morphogenetic movements of the epiblast that begin well before primitive
streak
formation (Gräper, 1929; Vakaet,
1970; Izpisua-Belmonte et al., 1993; Callebaut et al., 1999b; Foley et
al.,
2000).
At stage X, future “dorsal” tissues
(prospective
organizer and its derivatives: endoderm, prechordal mesoderm,
notochord) are
found just central to and adjacent to Koller’s sickle (Izpisua-Belmonte et al., 1993;
Hatada and Stern, 1994;
Bachvarova et al., 1998; Streit et al., 2000).
These territories quickly move towards the center of
the blastoderm, and gradually become replaced in their original
position by
more lateral regions of epiblast (progressively more “ventral” fates
like
somite, intermediate mesoderm, etc.) (see
supplementary movies: {Weijer.avi}
and {normal.avi}). Therefore at stage X the posterior margin of the
area
pellucida contains a bilateral gradation of dorsal-to-ventral fates,
dorsal at
the posterior mid-point – subsequent movements “fold” this arrangement
into a
posterior midline presaging the future primitive streak,
so that the most
dorsal fates become located most anteriorly along this line (see
below). These
movements comprise convergence of epiblast towards the posterior mid-point and
extension along the midline, but do not seem to occur by the process
normally
called “convergent extension” in that it is not
accompanied by significant cell shape changes. The combination of
posterior
midpoint convergence and midline extension resembles a Polish dance
(“Polonaise”) (see supplementary movies: {movie1} and {movie3}), the
name given by Gräper (Gräper, 1929)
to these epiblast movements after his remarkable
stereo-pair time-lapse films of labeled embryos, made as early as 1926.

It
is truly remarkable that cells can move horizontally within a
relatively tight
epithelium, the epiblast. The mechanics of such migration, including
the degree
to which it is truly “active” (rather than a consequence of mechanical
propagation of a remote event like cell loss
through ingression), is not yet
understood. However, recent observations of living, Bodipy-ceramide
chick
embryos using two-photon microscopy have started to reveal that
individual
cells within the epithelium and translocate by “bobbing” up and down,
as if
each individual cell is a foot in a giant millipede (O. Voiculescu,
I.-J. Lau,
F. Bertocchini and C.D. Stern, unpublished observations).

We
have already described briefly above the movements in the lower layer (Waddington, 1932; Spratt and Haas, 1960b;
Vakaet, 1970; Rosenquist, 1972; Stern and Ireland, 1981; Stern, 1990;
Bakst et
al., 1997; Callebaut et al., 1997a; Bachvarova et al., 1998; Foley et
al.,
2000; Bertocchini and Stern, 2002).
Essentially the hypoblast expands as the islands
fuse from posterior to anterior, and the newly-formed hypoblast sheet
is then
displaced further anteriorly by the incoming endoblast (see
supplementary movie
{movie1} and
animation {movie2}),.
The speed at which
the hypoblast/endoblast layer spreads is similar to the midline
extension in
the epiblast (Hatada and Stern,
1994)
and recent experiments have shown that there is a
causal link: rotation of the deep layer generates a new set of
Polonaise
movements in the adjacent epiblast (Foley
et al., 2000),
although the mechanisms by which the two layers
communicate are unknown.

These
movements also deform Koller’s sickle, which appears to be subjected to
a large
amount of shear. Its anterior (centrally-facing) mid-point will later
migrate
anteriorly as the primitive streak forms, its lateral extremes converge
to the
midline, and the posterior aspect (facing the marginal zone) remains
posterior
and eventually becomes extraembryonic (Izpisua-Belmonte
et al., 1993; Bachvarova et al., 1998; Streit et al., 2000).

Cell interactions leading to primitive streak formation

The fact that
isolated fragments
of blastodiscs can spontaneously initiate axis formation (Lutz,
1949; Spratt and Haas, 1960a)
indicates that cell interactions, rather than
definitive determinants, must be involved. What are the signals, and
where do
they come from? Three main sources have been proposed: the hypoblast
and/or
endoblast, Koller’s sickle and the posterior marginal zone (PMZ).

The
idea that
the hypoblast/endoblast layer regulates the site of primitive streak
formation
comes from important experiments by Waddington (Waddington,
1932, 1933)
in which he demonstrated that rotation of the deep
layer (which he called “endoderm”) influences the orientation of the
primitive
streak. When it was rotated by 90o the streak arose from its
original site but developed a gradual bend. After 180o
rotation of
the hypoblast, a few embryos had formed an ectopic streak arising from
the
opposite (anterior) side. This led Waddington to suggest that the
hypoblast
layer induces the primitive streak (Waddington,
1933)
but he was cautious to avoid ruling out a
contribution from cell movements. Subsequent studies were more forceful
in
proposing induction by the hypoblast (Azar
and Eyal-Giladi, 1979, 1981; Mitrani and Eyal-Giladi, 1981; Azar and
Eyal-Giladi, 1983; Mitrani et al., 1983)
but without providing direct evidence with molecular
markers or markers for different cell populations. Later, two studies
repeated
Waddington’s original observations and highlighted the fact that since
90o
hypoblast rotations do not induce primitive streak formation from a new
site,
this is unlikely to be a true inductive event (Khaner,
1995; Foley et al., 2000).
Moreover, it was shown (Foley et al., 2000)
that the hypoblast layer influences the movements of
the overlying epiblast – when rotated, it initates a new set of
Polonaise
movements at 90o to the original. These compete with the
original
movements causing the streak to bend, but cells destined for different
tissue
types do not change their fates.

Recently,
a
new emphasis has been placed on the second component of the deep layer,
the
endoblast (Callebaut and Van Nueten,
1995; Callebaut et al., 1998b; Callebaut et al., 1999b; Callebaut et
al.,
2000b; Bertocchini and Stern, 2002).
Specifically (Bertocchini
and Stern, 2002),
it was shown that complete removal of the hypoblast
leads to the formation of multiple streaks at random positions,
suggesting that
the hypoblast emits an antagonist of axis formation. Analysis of
expression
patterns and misexpression experiments then suggested that Cerberus, a
Nodal
antagonist, is responsible. Cerberus is expressed in the
hypoblast but
not in the endoblast, which is consistent with the fact that the
primitive
streak starts to form precisely at the time when the hypoblast is
displaced
away from the posterior edge of the area pellucida by the incoming
endoblatst (Bertocchini and Stern, 2002).
Finally, it should be mentioned that the hypoblast
does have some inducing activity, which can be revealed by assessing
the
expression of several epiblast genes after grafting a hypoblast
ectopically:
the homeobox gene Not1/GNOT (Knezevic
and Mackem, 2001),
and the early “pre-neural” markers ERNI, Sox3
and Otx2 are induced transiently by grafts of the hypoblast to
ectopic
sites (Foley et al., 1997; Streit et
al., 2000)
(see Neural induction). The
induction of Not1/GNOT may be mediated by retinoids, while
induction of ERNI
and Sox3 is mediated by FGF. We do not yet know the factors
responsible
for inducing Otx2.

The
second component suggested as playing a role in primitive streak
initiation is
Koller’s sickle (Izpisua-Belmonte et
al., 1993; Callebaut and Van Nueten, 1994; Callebaut et al., 1997a;
Callebaut
et al., 1998b; Callebaut et al., 2003).
This structure has been said to give rise to the
endoblast (hence the alternative name of “sickle endoblast”) and has
even been
proposed to act as a passage for posterior marginal zone cells from the
epiblast to the lower layer (Azar
and Eyal-Giladi, 1979; Eyal-Giladi, 1997).
However higher resolution fate mapping using
different techniques has suggested instead that the sickle contributes
cells to
the primitive streak itself but not significantly to the endoblast (Izpisua-Belmonte et al., 1993; Bachvarova
et al., 1998).
Furthermore although grafts of the sickle can indeed
generate a second primitive streak upon transplantation, the extensive
cellular
contribution to the ectopic streak and particularly to definitive (gut)
endoderm cannot be dissociated from the inductive effect (Izpisua-Belmonte
et al., 1993; Bachvarova
et al., 1998).

The
third and final tissue involved in induction of primitive streak
formation is
the posterior marginal zone (PMZ) (Spratt
and Haas, 1960a; Azar and Eyal-Giladi, 1979; Eyal-Giladi and Khaner,
1989;
Khaner and Eyal-Giladi, 1989; Callebaut et al., 1997b; Bachvarova et
al., 1998;
Bachvarova, 1999; Skromne and Stern, 2001, 2002).
Even though its activity as a streak inducer has
been challenged (Callebaut et al.,
1997b; Callebaut et al., 1998b),
there is no question that when grafted into an
ectopic position the PMZ is able to induce the formation of a second
axis
without making a cellular contribution to it, as long as the host is
younger
than stage XI (Eyal-Giladi and
Khaner, 1989; Khaner and Eyal-Giladi, 1989; Bachvarova et al., 1998).
These properties of the PMZ have likened it to the
amphibian Nieuwkoop center (see Chapters XXX – Keller amphibian;
Lemaire
Siamois). And like the Nieuwkoop center, whose activity appears to
depend upon
the overlap of TGFβ and Wnt pathways, the inducing ability of the PMZ
can be
mimicked by misexpression of cVg1 in regions where Wnt8C is expressed (Seleiro et al.,
1996; Shah et al., 1997; Skromne and Stern,
2001, 2002)
(Fig. 5). Surprisingly however, unlike PMZ grafts,
misexpression of cVg1 in the anterior marginal zone will
generate a full
axis as late as stage XIII.

In
conclusion, all three tissues (hypoblast/endoblast, Koller’s sickle and
PMZ)
proposed to have axis inducing activity do indeed have the ability to
influence
primitive streak formation. However, their mechanisms of action and
relative
importance differ. The sickle has inducing ability but this is probably
only
because it contains some of the cells fated to form Hensen’s node (the
avian
organizer – see below). The earliest influences appear to come from the
PMZ,
where Vg1 and Wnt activities overlap. Vg1+Wnt induce expression of
Nodal in the
neighboring area pellucida epiblast, but Nodal can only act (presumably
to
induce mesendoderm) when the hypoblast has been displaced by the
incoming
endoblast.

In
addition to
Vg1+Wnt and Nodal, it is likely that FGFs (emanating from the hypoblast
and/or
Koller’s sickle) (Chapman et al.,
2002; Karabagli et al., 2002)
also play a role in primitive streak initiation
because inhibitors of FGF block this process (Mitrani
et al., 1990; Streit et al., 2000)
and because misexpression of FGF can generate an
ectopic streak (F. Bertocchini, I. Skromne and C.D. Stern, unpublished
observations). It is likely that FGF acts in concert with Nodal, as in
amphibians (Kimelman and Kirschner,
1987; Cornell and Kimmelman, 1994; LaBonne and Whitman, 1994; Latinkic
et al.,
1997).
Finally, BMP activity also regulates primitive
streak formation since ectopic expression of the antagonist Chordin
(but not
Noggin) is sufficient to induce a streak, even as late as stage 3, and
misexpression of BMP4 near the streak causes the streak to disappear (Streit and Stern, 1999).
Chordin is normally expressed in Koller’s sickle at
stages XI-XIV.

Several
important questions still remain unanswered. They include: what
positions Vg1
expression in the PMZ? What molecular mechanisms underlie regulation
when the
posterior half of the blastoderm is removed?

Primitive streak formation and elongation

We know surprisingly little about the cellular
details of
how the primitive streak forms and elongates. Time-lapse films (see
movie1, movie3)
show that the initial appearance of the streak is extremely rapid – the
embryo
goes from having no visible axial structures (stage XIV) to developing
a
triangular, dense streak (stage 2; Fig. 3) in about 30 min, suggesting
that
primitive streak initiation is accompanied by massive ingression.
However,
although the basement membrane under the epiblast does partially
dissolve
during streak formation, this early stage does not involve the loss of
epithelial
continuity of the epiblast in the region of the forming streak, which
happens
much later (stage 3+) (Vakaet,
1982; Andries et al., 1983; Sanders and Prasad, 1989; Harrisson et al.,
1991).
This suggests that the formation of the early,
triangular-shape streak is the result of rapid poly-ingression of
individual
cells through the basal lamina of the epiblast. This process may
therefore be
analogous to the formation of primary mesenchyme in echinoderms.

Ingression
of early, “pioneer” cells from the epiblast to the interior of the
embryo can
be seen starting as early as stage XII by staining either with the
HNK-1
antibody (Canning and Stern, 1988;
Stern and Canning, 1990; Canning et al., 2000; Mogi et al., 2000)
or for activity of the enzyme Acetylcholinesterase
(AChE) (Drews, 1975; Valinsky and
Loomis, 1984; Laasberg et al., 1986; Parodi and Falugi, 1989).
Indeed, the HNK-1 epitope is carried on a subunit of
AChE (Bon et al., 1987),
and AChE activity correlates very well with cell
ingression and invasiveness in a variety of species (Drews,
1975).
It is puzzling that HNK-1/AChE expression differs in
different strains of fowl; the salt-and-pepper expression in the
epiblast is
seen in the Rhode Island Red/Light Sussex cross-breed but not in the
more
inbred strain, White Leghorn. It also seems clear that HNK-1/AChE
expression
does not correlate completely with cells destined to form the primitive
streak (Cooke, 1993).
A few of these cells do ingress but do not
contribute to the streak (their fate remains unknown), other
HNK-1-postive
cells remain in the germ wall margin from where they contribute to the
lower
layer (Canning and Stern, 1988;
Stern and Canning, 1990; Cooke, 1993),
and yet others do ingress to the primitive streak
but later seem to disappear. Careful fate maps examining the origin of
cells
that will form the primitive streak have revealed that the definitive
primitive
streak is largely derived from a relatively small population of
epiblast cells
local to the site of streak formation and from Koller’s sickle (Bachvarova et al., 1998; Wei and Mikawa,
2000).

The early
triangular streak (stage 2) is made up of a dense accumulation of
middle layer
cells between epiblast and endoblast (Fig. 3); however it rapidly
straightens,
to become a mesenchymal rod of parallel sides (stage 3). At this stage
there is
still no groove in the overlying epiblast and the basement membrane is
largely
intact. Soon afterwards however two processes take place more or less
simultaneously (Vakaet, 1970):
the appearance of a longitudinal groove in the
epiblast overlying the streak and the start of lateral migration of the
mesenchyme of the streak, at right angles to the axis of the streak, to
establish the lateral plate. These processes define stage 3+.
Since
grafts of early (stage 3) streak to a new area can generate an ectopic
streak
containing an epiblast groove (Vakaet,
1973),
and by analogy to the interactions between primary
and secondary mesenchyme in echinoderms, it seems likely that the early
streak
cells induce the formation of a groove (and subsequent invagination) in
the
overlying epiblast. The signals that mediate this interaction are
unknown but
FGF and/or Chordin are likely candidates since both can induce a streak
at
stage 3 (see above).

Stage 4 is marked by
the
appearance of a distinct bulge at the tip of the streak, encompassing
all three
layers – this is Hensen’s node (Hensen,
1876)
(see below). We consider this to be the last phase of
the “gastrula stage” in avian embryos – shortly afterwards (stage 4+)
a small triangular mass of cells starts to protrude anteriorly from the
node
(the emerging tip of the head process, which contains precursors for
the
prechordal mesendoderm – see below). At this time the future neural
plate
starts to become morphologically and molecularly (Sox2-positive)
distinct, and stage 4+ can therefore be considered the
beginning of
the “neurula stage”.

We also
know virtually nothing about the mechanics of primitive streak
elongation.
Time-lapse films reveal that less than 2 hours elapse between the early
short
streak at stage 2 and the almost fully elongated (1.5mm long) stage 3
streak,
making it very unlikely (L. Bodenstein, unpublished computer
simulations) that
cell division alone is the main force driving this elongation (Wei and Mikawa, 2000).
Streak elongation most likely involves a process of
cell reorganization and changes in cell shape similar to those seen in
amphibian convergent-extension.

Hensen’s node

The function of
Hensen’s node
(the avian organizer) will be described elsewhere (see Neural induction). Here
we will concentrate on the origin, maintenance and subdivision of the
node into
different cellular territories.

Various
fate mapping techniques have established that Hensen’s node arises from
two
distinct populations of cells (Izpisua-Belmonte
et al., 1993; Hatada and Stern, 1994; Bachvarova et al., 1998; Streit
et al.,
2000; Lawson and Schoenwolf, 2001).
One cell population (called “posterior cells” by
Streit et al., 2000) resides deep to the epiblast, at the midpoint of
Koller’s
sickle from stage X. It remains in this position until the primitive
streak
starts to form (stage 2), and then moves anteriorly with the tip of the
advancing streak. The second population (called “central cells” by
Streit et
al., 2000) resides in the epiblast during these early stages – at stage
X it is
found immediately adjacent to the first population (in the Nodal-expressing
territory; see Fig. 5 at stage X). However, the Polonaise movements
almost
immediately move these cells to the middle of the blastoderm, when
these
movements stop (stage XIII). As the primitive streak elongates, the
posterior
cells soon regain contact with the central cells (stages 3-3+).
At
this time, a morphological node forms (stage 4) and this is accompanied
by the
acquisition of expression of Sonic hedgehog (Shh) (Levin et al., 1995).

Neither
the posterior nor the central cells possess full neural inducing
ability by
themselves (although posterior cells can induce transient expression of
the
pre-neural genes Sox3 and ERNI), but acquire this
ability when
combined (Streit et al., 2000).
When transplanted ectopically into the area pellucida
of a host embryo, posterior cells can induce neighboring epiblast cells
to
acquire expression of goosecoid, a marker of the organizer (Izpisua-Belmonte et al., 1993).
These findings suggest that during their migration
anteriorly from stages 2-3, the posterior cells recruit adjacent
epiblast cells
to form part of the organizer.

The
cellular composition of Hensen’s node remains dynamic throughout the
early
stages of development: even after stage 3+, neighboring
epiblast
cells migrate to the node, acquire the expression of organizer markers
and
later migrate out again to emerge in the underlying layers as endoderm,
notochord, prechordal mesendoderm or medial somites (Joubin
and Stern, 1999).
Inducing signals from within the streak (again,
Vg1+Wnt, perhaps Nodal) and inhibitory signals from the node itself
(ADMP) and
from surrounding regions of the blastoderm (BMPs) form a complex
network
regulating the spatial and temporal expression of node markers
including Chordin,
Goosecoid, Shh, Not1, HNF3β and others (Joubin and Stern, 1999).
These results account for the fact that primitive
streak stage embryos from which the node has been extirpated can
generate a new
node (Grabowski, 1956; Psychoyos and
Stern, 1996b; Joubin and Stern, 1999; Yuan and Schoenwolf, 1999).

Despite
the
dynamic composition of the node at these stages, single cell lineage
analysis
has suggested that the node also contains a small population of
resident cells
with stem-cell characteristics (Selleck
and Stern, 1991; Selleck and Stern, 1992b).
It was proposed that when these cells divide, one
daughter remains in the node while the other leaves to contribute to
notochord
and/or medial somite (Selleck and
Stern, 1992b; Stern et al., 1992).

The
node is
not a uniform structure, either molecularly or by tissue fate. At a
molecular
level, it displays left-right asymmetry of expression of a number of
genes. The
earliest of these are Activin receptor IIA (more likely to be a
receptor
for Nodal) which is expressed on the right and the transcription factor
HNF3β
which is expressed on the left (Levin
et al., 1995; Stern et al., 1995),
from stage 3+. By stage 4-4+,
while the node starts to develop slight morphological asymmetry, Shh
appears on the left, FGF8 on the right and Nodal just
to the left
of the node (Levin et al., 1995;
Dathe et al., 2002).
Different regions of the node also give rise
preferentially to different structures (Fig. 6), although the
boundaries
between these fates are not sharp. Specifically, the tip of the node
contains
mainly prospective notochord, prechordal mesendoderm and floor plate
cells, the
sides and posterior aspect have mainly prospective medial somite and
endodermal
precursors (Selleck and Stern, 1991).
Transplantation experiments have revealed that the
prospective notochord region contains cells that are already committed
to this
fate while the lateral regions are more plastic (Selleck
and Stern, 1992a),
but that all regions of the node are
indistinguishable in their ability to induce and pattern neural tissue (Storey et al., 1995).

Establishment and subdivision of embryonic endoderm and mesoderm

The study of endoderm
formation
in avian embryos, as in many other species, has been hindered
considerably by
the lack of any exclusive, permanent markers for the endoderm lineage.
This is
even more inconvenient because the endoblast (see above) also lacks
specific
molecular markers, which makes it very difficult to distinguish these
two
neighboring tissues except that the endoblast contains typical
intracellular
inclusions which can be seen under phase contrast in explanted tissues (Stern and Ireland, 1981).
It was not until 1953 that Bellairs first recognized
that the definitive endoderm is derived from the epiblast via the
primitive
streak (Bellairs, 1953a, b, 1955,
1957)
rather than from the hypoblast layer as was
previously thought. The endoderm probably
starts to insinuate itself into the lower layer at the early primitive
streak
stage (stage 2), and this insertion process ends by stage 4 (Vakaet, 1962; Nicolet, 1965; Modak, 1966;
Gallera and Nicolet, 1969; Nicolet, 1970; Selleck and Stern, 1991).
We are still ignorant about the signals that induce
and pattern the endoderm, from where they arise and at what stage, but
based on
studies in other species it seems likely that Nodal will turn out to
play a
major role.

By
the time the endoderm inserts into the deep layer (stage 3+-4),
the
original hypoblast cells have become confined to the most anterior part
of the
blastoderm, a region called the “germinal crescent” because it also
contains
the primordial germ cells (Ginsburg
and Eyal-Giladi, 1986, 1987, 1989; Ginsburg et al., 1989; Tsunekawa et
al.,
2000).
Since the surface of the hypoblast is greater than
that of the germinal crescent, the tissue often develops blister-like
projections extending ventrally from the surface of the epiblast. The
fate of
the hypoblast cells after this stage has not been examined thoroughly
but it is
generally assumed that they contribute to the stalk of the yolk sac. It
is
equally likely however that a large proportion of hypoblast cells
undergo
apoptosis, since TUNEL staining at this stage shows heavy labeling in
the
hypoblast of the germinal crescent (A. Gibson and C.D. Stern,
unpublished
observations).

The
bulk of the middle layer of the stage 3+ primitive streak
will give
rise to mesoderm: the notochord in the midline with prechordal mesoderm
at its
tip, the somites, intermediate mesoderm (prospective mesonephric kidney
and its
duct), heart, lateral plate mesoderm (which includes both embryonic and
extraembryonic components). It is important to recognize that the long
axis of
the primitive streak does not correspond to the future head-tail axis
of the
embryo but rather to the future dorsoventral axis of the mesoderm:
anterior
streak (node) gives rise to the most dorsal/axial structures, with more
ventral
(lateral) structures arising from progressively more posterior streak
positions
(Schoenwolf et al., 1992; Psychoyos
and Stern, 1996a; Sawada and Aoyama, 1999; Freitas et al., 2001;
Lopez-Sanchez
et al., 2001).
This can be understood most easily by looking at the
patterns of cell migration from the streak (Figs. 4, 6), and the same
relationship is seen in the mouse primitive streak.

It
is likely
that the major player in imparting specific dorsoventral identity to
prospective mesoderm is BMP signaling. The node, which emits BMP
antagonists,
can transform lateral plate mesoderm into somitic mesoderm (Nicolet, 1968)
and this has been shown to be mimicked by Noggin (but
not Chordin) (Streit and Stern,
1999).
Since Noggin is not expressed in the chick until
about stage 4+, it is likely that somite identity is not
fixed until
after this stage. Indeed, competence for lateral-to-medial
transformation and
vice-versa remain until at least the early somite stage (Tonegawa
et al., 1997; Streit and Stern, 1999; James and
Schultheiss, 2003).

Ingression
from the epiblast to the deeper layers to form endoderm and the most
medial
(axial) mesoderm ends around the end of stage 4 (Vakaet,
1962; Nicolet, 1965; Modak, 1966; Gallera and Nicolet,
1969; Nicolet, 1970; Selleck and Stern, 1991; Joubin and Stern, 1999).
A recent study has identified a zinc finger
transcriptional activator, Churchill, which regulates the
cessation of
ingression at the primitive streak by activating Sip1, an
antagonist of Brachyury (Sheng et al., 2003).
This is described in more detail under Neural
induction; here we will only point out that expression and
activites of Churchill and Sip1 regulate the transition
from the
end of gastrular ingression to the start of neurulation opposite the
anterior
levels of the primitive streak.

Time-lapse
films show that formation of the head process (the name given to the
cranial
portion of the notochord, rostral to the future level of the otic
vesicle)
begins at stage 4+ by forward migration of cells from the
node (Spratt, 1947; Bellairs, 1953b).
After a short delay (to the end of stage 5) these movements
stop and the primitive streak starts to regress (see below), which
continues to
extend the notochord caudally. Elongation of the notochord appears to
include
both a process of convergent-extension (as in amphibians and fish) and
the gradual deposition of progeny
from resident stem cells (see above), but the major ingression
movements from
the epiblast opposite the anterior primitive streak have ceased by this
stage.
At more posterior levels, however, ingression to form lateral mesoderm
continues for some time.

The
emigration
of prospective somite and lateral plate mesoderm from the primitive
streak is
controlled by chemorepulsion by FGF (perhaps FGF8) expressed in the
streak (Yang et al., 2002).
Yang et al. also proposed
that after emerging from the streak, prospective somite tissue is then
attracted back to the midline, specifically to FGF4 expressed in the
notochord.
However, since the entire embryo elongates and narrows at this stage it
is
difficult to determine whether the migration of somitic mesoderm
towards the
midline is as active a process as was proposed (Easton
et al., 1990).
Furthermore, embryos lacking a notochord make a
midline row of somites underlying the neural tube, raising the question
of what
would attract cells to the midline if this model is indeed correct (Stern and Bellairs, 1984).

Ending gastrulation and regression of the primitive streak

The main period of
gastrulation
is characterized by massive movement of epiblast into the primitive
streak to
generate mesoderm and endoderm. These movements gradually stop from
stages 4-4+
at the most anterior levels of the streak (prospective notochord and
medial
somite), and progressively more caudally. As mentioned above, the end
of
ingression through the anterior streak is regulated by Churchill
and Sip1 (Sheng et al., 2003).
Soon after this (between stages 5-6), the primitive
streak starts to regress (see supplementary animation
{ChickGastula_animation.avi}),.

Several
studies have attempted to establish the main cellular forces driving
regression
of the primitive streak. The earliest (Spratt,
1947)
made the important discovery that shortening of the
streak is predominantly a morphological change, rather than a migration
of node
cells. However, convergent-extension also plays a major role in the
process as
mentioned earlier (Spratt, 1947;
Bellairs, 1963; Lepori, 1966; Stern and Bellairs, 1984; Schoenwolf et
al.,
1992; Catala et al., 1996; Colas and Schoenwolf, 2001).
We still know nothing, however, about the signals
that regulate the timing, the speed or the specific changes in cell
behavior
that control regression.

The tail bud – a continuation of gastrulation?

While regression
continues, the
deposition of axial and paraxial mesoderm continue as the whole embryo
narrows
and elongates caudally to generate the tail bud (Sanders
et al., 1986; Catala et al., 1996; Knezevic et al.,
1998; Charrier et al., 2002).
It was therefore proposed that the tail bud is a
continuation of the process of gastrulation (Knezevic
et al., 1998).
While it is true that several processes
characteristic of gastrulation do continue in the regressing streak and
later
in the forming tail bud, other critical processes do not. Specifically,
massive
ingression of epiblast to form axial tissues (notochord and somites)
has ceased
(except perhaps at the most caudal end), the formation of new endoderm
from the
streak has also ended, and regression of the streak is accompanied by
cell
depletion from this structure. Furthermore the node starts to lose its
neural
inducing ability just after stage 4 (Dias
and Schoenwolf, 1990; Storey et al., 1995)
(Neural Induction). Together
with the fact that the neural plate starts to elevate at about stage 4+ (Bancroft and Bellairs, 1975),
we consider that the end of gastrulation (as a
stage) occurs between stages 4 and 4+.

Azar, Y. and
Eyal-Giladi, H.
(1981). Interaction of epiblast and hypoblast in the formation of the
primitive
streak and the embryonic axis in chick, as revealed by
hypoblast-rotation
experiments. J Embryol Exp Morphol61,
133-144.

Bancroft, M. and
Bellairs, R.
(1974). The onset of differentiation in the epiblast of the chick
blastoderm
(SEM and TEM). Cell Tissue Res155,
399-418.

Bancroft, M. and
Bellairs, R.
(1975). Differentiation of the neural plate and neural tube in the
young chick
embryo. A study by scanning and transmission electron microscopy. Anat Embryol (Berl)147, 309-335.

Bellairs, R. (1953a).
Studies on
the development of the foregut in the chick blastoderm. 1. The
presumptive
foregut area. J. Embryol. exp. Morph.1, 115-124.

Bellairs, R. (1953b).
Studies on
the development of the foregut in the chick blastoderm. 2. The
morphogenetic
movements. J. Embryol. exp. Morph.1,
369-385.

Bellairs, R. (1955).
Studies on
the development of the foregut in the chick embryo. 3. The role of
mitosis. J. Embryol. exp. Morph.3,
242-250.

Bellairs, R. (1957).
Studies on
the development of the foregut in the chick embryo. 4. Mesodermal
induction and
mitosis. J. Embryol. exp. Morph.5,
340-350.

Bellairs, R. (1963).
The
development of somites in the chick embryo. J.
Embryol. exp. Morph.11,
697–714.

Eyal-Giladi, H. and
Kochav, S.
(1976). From cleavage to primitive streak formation: a complementary
normal
table and a new look at the first stages of the development of the
chick. I.
General morphology. Dev Biol49,
321-337.

Fabian, B. and
Eyal-Giladi, H.
(1981). A SEM study of cell shedding during the formation of the area
pellucida
in the chick embryo. J Embryol Exp
Morphol64, 11-22.

Ginsburg, M. and
Eyal-Giladi, H.
(1986). Temporal and spatial aspects of the gradual migration of
primordial
germ cells from the epiblast into the germinal crescent in the avian
embryo. J Embryol Exp Morphol95,
53-71.

Ginsburg, M. and
Eyal-Giladi, H.
(1987). Primordial germ cells of the young chick blastoderm originate
from the
central zone of the area pellucida irrespective of the embryo-forming
process. Development101, 209-219.

Kochav, S., Ginsburg,
M. and
Eyal-Giladi, H. (1980). From cleavage to primitive streak formation: a
complementary normal table and a new look at the first stages of the
development of the chick. II. Microscopic anatomy and cell population
dynamics.
Dev Biol79, 296-308.

Yuan, S. and
Schoenwolf, G. C.
(1999). Reconstitution of the organizer is both sufficient and required
to re-
establish a fully patterned body plan in avian embryos. Development126,
2461-2473.

FIGURE LEGENDS

Fig. 1.
Formation of the
hens’ egg and its descent along the maternal oviduct. From (Duval, 1889).

Fig. 2.
Cleavage in the
chick embryo. A. A section through the yolk reveals concentric
rings (3)
of dense (darker) and white (lighter) yolk. Under the blastoderm (1), a
sub-blastodermic space filled with white yolk forms a funnel (the
latebra) that
extends deep into the center of the yolk mass, where it forms a small
cavity,
the Nucleus of Pander (2). B. Cleavage in the chick embryo is
meroblastic: the cleavage planes open into the surrounding yolk mass. C.
A section through the blastoderm and the surrounding yolk reveals the
subgerminal cavity and latebra. All three figures dapted from (Duval, 1889).

Fig. 4.
Summary fate maps
of the epiblast and cell movement patterns at different stages. The two
diagrams on the left hand column show the major movements in the
epiblast:
Polonaise movements before primitive streak formation, and convergence
of the
epiblast to the streak (which is strongest posteriorly) during
gastrulation.
The middle column of diagrams summarize the locations of territories of
cells
that give rise to different mesodermal tissues and the gut endoderm.
The
rightmost column summarizes the locations of subdivisions of the neural
plate.
The dashed line at stage XI indicates the most anterior extent of
spread of the
hypoblast layer at this stage, and the dashed outline at stage 3 is the
profile
of the primitive streak.

Fig. 5.
Molecular
interactions implicated in the initiation of primitive streak
formation, shown
at three successive stages (indicated on the left), in sections (left
column)
and in whole mounts (right). At stage X, Vg1 (red; expressed in the
posterior
marginal zone) cooperates with Wnt8C (blue; expressed throughout the
marginal
zone) to induce Nodal (bright green) in the neighboring
epiblast of the
area pellucida. However, Nodal cannot act further because it is
inhibited by
Cerberus (black) produced by the underlying hypoblast (stage XII).
Shortly
before primitive streak formation (stages XIV-2), the displacement of
the
hypoblast by the non-Cerberus-expressing endoblast allows Nodal
signaling to
act. Nodal, in cooperation with FGF (light brown; emanating from the
hypoblast
and from Koller’s sickle) and Chordin (dark green; produced by Koller’s
sickle)
then induce ingression of cells from the epiblast to form the primitive
streak.
(Based on data from several sources, mainly (Skromne
and Stern, 2001; Bertocchini and Stern, 2002).

Fig. 6. Fate
maps of the
primitive streak and Hensen’s node. A. Morphology of the
anterior tip of
the primitive streak at different stages: stage 3 (no groove, parallel
sides),
3+ (groove, parallel sides), 4 (distinct node), 4+
(incipient
head process, elongated pit). B. Fates and movement patterns of
mesoderm
emerging from different portions of the streak at stage 4. Note that
the
anterior-posterior axis of the streak corresponds not to the head-tail
axis of
the embryo but rather to the mediolateral (axial-lateral, or
dorsoventral) axis
of the mesodermal organs. Based on data from several sources, mainly (Selleck and Stern, 1991; Psychoyos and
Stern, 1996a).

What is neural
induction?

Embryonic induction
has been
defined by Gurdon as “... an interaction between one (inducing)
tissue and
another (responding) tissue, as a result of which the responding tissue
undergoes a change in its direction of differentiation” (Gurdon, 1987).
Neural induction is therefore the process by which
cells acquire a neural fate in response to appropriate signals during
development or after embryonic manipulations that bring two dissimilar
cell
types together. During normal vertebrate development, neural induction
is generally
believed to occur around the time of gastrulation, directed at least in
part by
signals emanating from a special region of the embryo: “the organizer”.
The
organizer resides in the embryonic shield of teleosts, the dorsal lip
of the
blastopore in amphibians and the tip of the primitive streak (Hensen’s
node) in
amniotes. This Chapter takes an historical approach to trace the
development of
our understanding of this process at the cellular and molecular levels.

Early history

The concept of
induction originates
in von Baer’s work (von Baer, 1828)
and was further developed at the turn of the 20th
Century notably by Curt Herbst (Oppenheimer,
1991).
But the concept of neural induction really

evolved
from the pioneering experiments of Warren Lewis (Lewis,
1907)
and the better known work of Hans Spemann and Hilde
Mangold (Spemann and Mangold, 1924;
Hamburger, 1988; De Robertis and Aréchaga, 2001)
(Figs. 1, 2). As part of an effort to resolve an
on-going controversy about whether embryos are “regulative” or
“mosaic”, Lewis
found that transplantation of the dorsal lip of the blastopore of Rana
to an ectopic position caused a second axis to form. However, he
interpreted
this as self-differentiation of the graft and it was not until
Spemann’s use of
interspecies grafts between three differently pigmented species of
newts (Triturus
taeniatus, T. cristatus and T. alpestris) (Spemann, 1921; Spemann and Mangold, 1924),
allowing the cells of the donor and host to be
distinguished, that it could be clearly concluded that this was an
example of
an inductive interaction. Spemann and Mangold termed the dorsal lip of
the
blastopore “the organizer”, because it could direct the formation of a
coherently
organized, ectopic axis from cells whose fate was other than to form
axial
structures.

It
took only a
few years for these findings to be extended to other vertebrates,
including
amniotes: first to avian species (chick and duck) (Hunt,
1929; Waddington, 1930, 1932; Waddington, 1933b)
and shortly afterwards to mammalian embryos (rabbit),
by interspecies grafts in all combinations (Waddington,
1932, 1934; Waddington, 1936, 1937).
In all these cases the primitive streak, and
specifically Hensen’s node at its anterior end, were found to contain
the
“organizer activity”.

The
ability of
the organizer to induce a nervous system is coupled with its ability to
pattern
the induced structures, the property that led to its name.
Anteroposterior
patterning is discussed elsewhere in this book (Fraser and Stern,
2004); suffice
it to say here that several models have been proposed to account for
this
activity of the organizer, the main ones being the “head/trunk/tail”
model most
clearly formulated by Otto Mangold (Mangold,
1933),
which proposes the existence of separate inducing
activities for the head, trunk and tail portions of the axis, and the
“activation/transformation” model of Nieukwoop (Nieuwkoop
et al., 1952; Nieuwkoop and Nigtevecht, 1954),
which proposes that the nervous system that is
initially induced is of “anterior” (forebrain) character and that later
signals
“transform” parts of it to more caudal fates. Currently there is
evidence both
for and against both opposing models and the issue has not yet been
fully
resolved (see Stern, 2001).
The rest of this essay will concentrate on neural
induction proper – the cellular and molecular mechanisms leading to the
specification of neural fate regardless of its rostrocaudal character.

Seven fruitless
decades

Following the
identification of
the organizer and of neural induction, the hunt began for the
“organizing
principles”. Spemann himself favored a vitalistic explanation, while
several
laboratories (most notably those of Holtfreter and O. Mangold and later
Tiedemann and Grunz in Germany, Toivonen and Saxén in Finland, Dorothy and Joseph
Needham and Waddington in England, Nakamura and Yamada in Japan and
Brachet in
Belgium) embarked on trying to identify a chemical inducer (Holtfreter, 1933; Waddington, 1933a;
Holtfreter, 1934; Needham et al., 1934; Spemann, 1938; Toivonen, 1938;
Chuang,
1939, 1940; Toivonen, 1940; Waddington, 1940; Holtfreter, 1945; Saxén
and Toivonen,
1962; Toivonen et al., 1975; Rollhauser-ter Horst, 1977b, a; Saxen,
1980; Chen
and Solursh, 1992;
reviewed in Nakamura
and Toivonen, 1978).
Early indications for a steroid, then for various
protein or RNA extracts, led to transient flurries of excitement, which
quickly
waned as a result of the discovery that numerous “heterologous”, or
non-specific inducers (including killed organizers, high or low pH,
alcohol,
histological dyes, …) were just as effective as an organizer graft in
inducing
a second axis in amphibians. Essentially no progress was made until
well into
the 1990s.

A turning point:
BMP antagonism and the “default
model”

Several
seemingly unrelated observations gradually led to a new concept,
commonly known
as “the default model” for neural induction (Hemmati-Brivanlou
and Melton, 1997)
(Fig. 3). First, several groups had observed that in
amphibians, dissociation of
gastrula-stage animal caps into single cells for a short time before
reaggregating them again leads to the formation of neural tissue (Born et al., 1989; Godsave and Slack,
1989; Grunz and Tacke, 1989; Sato and Sargent, 1989; Saint-Jeannet et
al.,
1990).
A few years later, it was found that misexpression
of a dominant-negative “activin”-receptor (it was later discovered that
this
construct inhibits several TGFβ-related factors) into Xenopus
embryos
not only blocks mesoderm formation but also generates ectopic neural
tissue (Hemmati-Brivanlou and Melton, 1992, 1994).
At about the same time, it was discovered that BMP4
is a ventralizing factor in Xenopus (Dale
et al., 1992; Jones et al., 1992).
Several of these authors speculated that neural
induction might be induced by removal of some inhibitory substance (Hemmati-Brivanlou and Melton, 1994),
but direct evidence was still lacking.

Together, these findings led to the
“default model” (Hemmati-Brivanlou
and Melton, 1997),
which proposes that cells within the ectoderm layer
of the frog gastrula have an autonomous tendency to differentiate into
neural
tissue. This tendency is inhibited by bone morphogenetic proteins - in
particular, BMP4, which acts as an epidermal inducer (Fig. 3).

Consistent with this model (Fig. 4),
neuralization does not occur after dissociation of animal caps obtained
from
embryos previously injected with RNA encoding effectors of BMP4 (Msx1, Smad1 or Smad5; Suzuki et al.,
1997a; Suzuki et al., 1997b; Wilson et al., 1997),
consistent with the view that the neural pathway is
inhibited by an endogenous BMP-like activity. Moreover, the expression
pattern
of BMP4 in Xenopus conforms to its
proposed anti-neural function: in the early
gastrula, BMP4 transcripts are widely
expressed in the entire ectoderm and then clear from the future neural
plate at
the time when the organizer appears (Fainsod
et al., 1994).
Transcription of BMP
RNA is maintained by the activity of BMP protein (Biehs
et al., 1996),
which accou

nts
for the disappearance of BMP4 and -7
expression from the vicinity of the organizer (which secretes
BMP inhibitors) at the gastrula stage (Fainsod
et al., 1994; Hawley et al., 1995).

The model is further supported by
the effects of treatments that inhibit the BMP signaling pathway.
Animal caps
cut from embryos injected with either RNA encoding dominant-negative
receptors
that bind BMPs (Hemmati-Brivanlou
and Melton, 1994; Xu et al., 1995),
or non-cleavable forms of BMP4 or -7 (Hawley et al., 1995),
or antisense BMP4
RNA (Sasai et al., 1995)
adopt a neural fate instead of epidermis (Fig. 4).
Finally, Chordin and Noggin protein can neuralize isolated animal caps
(provided that these have been exposed briefly to low Ca++/Mg++-medium
– effectively a partial dissociation, although the rationale given for
this is
that it helps the protein penetrate between the cells).

In addition to its role in neural
induction, the organizer can also pattern the mesoderm at the gastrula
stage
(“dorsalization”). This activity can also be
attributed to BMP inhibition. BMPs can modify dorsal mesoderm to give
ventral
cell types (Dale et al., 1992;
Fainsod et al., 1994; Jones et al., 1996),
while their inhibitors can generate notochord and
muscle from ventral mesoderm (Smith
et al., 1993; Sasai et al., 1994; Tonegawa et al., 1997; Tonegawa and
Takahashi, 1998; Streit and Stern, 1999b).
BMP inhibitors can also regulate the dorsoventral
polarity of the whole embryo before gastrulation. For example, UV
irradiated
embryos lack dorsoventral polarity and fail to gastrulate, but can be
rescued
fully by injection of RNA encoding any of the BMP inhibitors: the
blastopore
(dorsal) will form close to the site of injection (Smith
and Harland, 1992; Sasai et al., 1994).

Finally,
a recent study by the De Robertis lab demonstrated that organizer
activity, or
at least dorsalization, requires functional Chordin (Oelgeschlager
et al., 2003).
Together, these findings provide compelling evidence
that BMPs and their modulation by endogenous inhibitors are involved in
the
activities of the organizer, including the establishment of neural and
non-neural domains in Xenopus. This
model is very attractive both because of its simplicity and also
because it
provides the first truly coherent model to explain neural induction
since the
discovery of the organizer by Spemann and Mangold.

To
some extent this explains some contradictory findings. For example, the
Wnt
pathway has been reported to act as a neural inducer by some (Sokol et al., 1995; Baker et al., 1999;
Wessely et al., 2001)
but to inhibit neural induction by others (Wilson et
al., 2001).
The difference could be accounted for if at early
stages Wnt dorsalizes the embryo (inducing tissues with organizer
properties),
while at later stages Wnt activity somehow antagonizes other signals
from the
organizer (Bainter et al., 2001;
Wilson and Edlund, 2001).
It becomes important to use reagents that work in a
cell-autonomous manner and to express them in a stage- and
position-controlled
way (as appropriate to the specific inductive event being studied).

A
more complex literature surrounds the role of FGF signaling in neural
induction. A first study implicated this pathway indirectly when
Suramin (which
inhibits FGF among other related proteins) was found to block neural
induction
in Xenopus (Grunz, 1992).
Later, several labs found that FGF can induce neural
tissue under certain circumstances (Lamb
and Harland, 1995; Rodriguez-Gallardo et al., 1997; Alvarez et al.,
1998;
Barnett et al., 1998; Storey et al., 1998; Hongo et al., 1999;
Hardcastle et
al., 2000; Ishimura et al., 2000; Wilson et al., 2000; Kim and Nishida,
2001;
Hudson et al., 2003),
while other studies suggested that FGF is not a
sufficient signal for neural induction (Amaya
et al., 1991; Cox and Hemmati-Brivanlou, 1995; Kroll and Amaya, 1996;
Holowacz
and Sokol, 1999; Ribisi et al., 2000; Pownall et al., 2003).
One explanation for this discrepancy is the
observation that different FGF receptors are required to mediate
different
activities of FGF: FGFR1 is required for the mesoderm-inducing function
of FGF,
while FGFR4 appears to mediate its role in neuralization (Hardcastle
et al., 2000; Umbhauer et al.,
2000).
The involvement of FGF signals in neural induction
will be discussed further below.

FGF, Wnt and BMPs
in neural induction

Despite the obvious
attraction of
the default model, several observations in different organisms do not
fit its
proposals so neatly. In Xenopus, inhibition of FGF signaling by
a
dominant-negative version of the FGF-receptor-1 (XFD) blocks the
neuralizing
activity of both Noggin and Chordin (Launay
et al., 1996; Sasai et al., 1996).
Furthermore, mere cutting of the animal cap
activates MAP kinase by phosphorylation, at least transiently (LaBonne and Whitman, 1997),
which could explain the finding made by several labs
that “control” animal caps express markers for the cement gland (Fig. 4). These
observations suggest that FGF signaling is required for neural
induction in
addition to BMP inhibition.

In
chick
(Figs. 5-6), the patterns of expression of components of the BMP
pathway do not
agree with the model: Chordin continues to be expressed in organizer at
stages
when this has lost its neural inducing activity, and Noggin and
Follistatin are
not expressed in the organizer at the appropriate stages at all, while
BMP4 and
BMP7 are expressed only weakly (if at all) in the ectoderm before
neural induction
begins, and their expression increases at the border of the neural
plate
starting from the end of gastrulation (stage 4) (Streit
et al., 1998).
Moreover, misexpression of Chordin or Noggin in
competent epiblast does not neuralize the epiblast (Streit
et al., 1998; Streit and Stern, 1999b; Linker et al.,
2004)
(Fig. 6) and dissociation of the epiblast leads to
differentiation of muscle rather than neurons (George-Weinstein
et al., 1996).
In zebrafish, neither Noggin nor Follistatin are
expressed in the organizer (Bauer et
al., 1998)
and Chordin (chordino) mutants,
although ventralized, still have a neural plate (Hammerschmidt
et al., 1996a; Hammerschmidt et al., 1996b;
Kishimoto
et al., 1997; Schulte-Merker et al., 1997; Bauer et al., 1998).
In mouse, BMP4 mutants are uninformative (the
embryos die too early with mesoderm and other generalized defects (Winnier et al., 1995),
but BMP2 and BMP7 mutants lack an
early neural phenotype (Dudley et
al., 1995; Zhang and Bradley, 1996)
and Chordin-, Noggin- and even Chordin-Noggin
double mutants have a respectable neural plate (Brunet
et al., 1998; McMahon et al., 1998; Bachiller et al.,
2000).
In the urochordate Ciona, FGF signaling through the
MEK pathway but not BMP inhibition appears to be responsible for neural
induction (Darras and Nishida, 2001;
Hudson and Lemaire, 2001; Kim and Nishida, 2001; Bertrand et al., 2003;
Hudson
et al., 2003).

FGF
signaling now appears to be a prerequisite for neural induction but
this step
occurs (or at least begins) very ear

ly,
before gastrulation (Streit et al., 2000; Wilson et
al., 2000).
However, FGF does not appear to be a sufficient or
direct neural inducer in vertebrates (Streit
et al., 2000).
Wilson and colleagues (Wilson et al., 2001)
suggested that FGF only shows neural inducing
activity when Wnt signaling is also blocked, and proposed that there
are two
divergent pathways both involving FGF: for “medial epiblast cells”
(prospective
neural plate), FGF signaling alone is sufficient to repress the BMP
pathway and
thus cause neuralization. For “lateral epiblast cells” (prospective
epidermis),
both FGF signaling and Wnt inhibition are required to block the BMP
pathway and
neuralize (Wilson and Edlund, 2001;
Wilson et al., 2001).
It has been proposed (Bainter et al., 2001; Wilson and
Edlund, 2001)
that the critical event involves regulation of BMP4
transcription. Most of these experiments have been conducted using
explants,
and in our own experiments in intact embryos we are unable to neuralize
competent epiblast by any combination of FGF, Wnt antagonists and/or
BMP
antagonists at any stage of development (Streit
et al., 2000; Linker et al., 2004).
Furthermore, we find that misexpression of the
intracellular BMP antagonist Smad6 in chick embryos is not
sufficient to
cause neuralization of competent epiblast even when combined with
secreted BMP
antagonists (Chordin and Noggin), FGF, secreted Wnt antagonists (NFz8,
Dkk1 and
crescent) and a multifunctional antagonist (Cerberus) (Linker
et al., 2004).
We therefore believe that not all the required
signals have been identified, and that although down-regulation of BMP
is
almost certainly involved in the specification of the neural plate,
this is not
a sufficient signal, even in combination with FGF and Wnt inhibition.

Looking upstream
from a critical promoter

To date, therefore,
we have not
yet arrived at a full understanding of the molecular signals that
trigger the
acquisition of neural fate by the ectoderm. One reason for this may be
the
diversity of approaches used in different “model” systems, and that
many of the
approaches used have not taken full account of the issue of
developmental
timing, which is particularly important when studying molecules with
multiple,
and sometimes opposing functions at different times. One might however
gain
further insight by changing the viewpoint to the promoter of a target
gene.
This has recently been attempted for the first time by analysis of the Sox2
promoter, which is a good marker for committed, developing neural plate
in the
chick (unlike the mouse where the early functions of Sox3 and Sox2
appear to have been exchanged). Kondoh and colleagues have identified
many
enhancers both upstream and downstream of the cSox2 gene, which
are
conserved in mouse and human (Uchikawa
et al., 2003).
Two of these enhancers, N1 and N2, appear to be
responsible for the onset of expression of Sox2 in the early
neural
plate – they contain binding sites for several identified transcription
factors, including a Sox-related protein (perhaps Sox3, which
in the
chick is expressed earlier in a similar domain to Sox2),
TCF/LEF (Wnt
pathway), homeodomain-containing proteins and an E-box sequence shown
to be a
target of Sip1/δEF1 (Verschueren et
al., 1999).
Sip1 was recently identified as a target of
the zinc finger protein Churchill, and morpholino-mediated
down-regulation of
Churchill function leads to loss of the neural plate, while
misexpression of Churchill
can confer or maintain the competence of epiblast to neural inducing
signals
from the node (Sheng et al., 2003)
(see Avian gastrulation).

A view from the
streak/blastopore

Churchill was
first
isolated from a molecular screen designed to identify genes that a

re
regulated
by 5 hours of signaling from a graft of the organizer, Hensen’s node,
in the
chick embryo (Sheng et al., 2003).
This was done because previous studies had revealed
that 5 hours’ exposure to a node are required for epiblast cells to
become
sensitive to Chordin misexpression (by maintaining Sox3
expression,
which is otherwise only transiently induced by a node graft) (Streit et al., 1998).
Churchill is expressed in the prospective
neural plate from the late gastrula stage and thereafter persists in
the
forming neural plate in a pattern similar to that of Sox2. Both
a node
graft and misexpression of FGF induce Churchill expression in
about 4
hours, as expected from the screen (Sheng
et al., 2003)
(Fig. 7). Churchill misexpression close to the
streak of the chick embryo or the blastopore of frog embryos causes
down-regulation of the mesodermal marker Brachyury; however,
Churchill
is a transcriptional activator, which suggested that one of its targets
may be
a repressor of Brachyury. A selection strategy and gel mobility
shift
assays identified the sequence CGGGRR as a binding target of Churchill,
and
analysis of the putative regulatory regions of Sip1 identified
numerous
occurrences of this sequence. Indeed Sip1 is expressed
identically to Churchill
and morpholino-knockdown of the latter causes loss of Sip1
expression (Sheng et al., 2003).
Sip1 is a good candidate to mediate the down-regulation
of Brachyury by Churchill, since it this is one of its known
functions (Verschueren et al., 1999; Lerchner et
al., 2000; Papin et al., 2002).

Misexpression
of Churchill near the primitive streak causes not only loss of Brachyury
but also a failure of cells to continue to ingress through the streak
to form
mesendoderm. Since Churchill begins to be expressed at about
the time
this ingression stops through the anterior primitive streak (see
Avian gastrulation), this raised the possibility
that one of its functions may
be to
end the process of gastrulation to keep some epiblast cells on the
surface.
This hypothesis is supported by the finding that morpholino
down-regulation of
Churchill at the late gastrula/early neurula stage causes cells to
continue to
ingress through the streak, and ectopic contribution to the mesoderm
rather
than neural plate. This effect can be rescued by co-electroporation of
either Churchill
or of its target Sip1 (Sheng
et al., 2003).
Thus, Churchill regulates the end of ingression
through the streak as well as the competence of the cells that express
it to
respond to neural inducing signals from the node, raising the
possibility that
this is a critical protein in the neural induction process, and
particularly in
regulating the transition from gastrulation to neurulation. This
finding drew
attention to the rather overlooked fact that at the gastrula stage, the
prospective mesoderm territory lies adjacent to the prospective neural
plate in
all vertebrate classes, and to the possibility that during normal
development
the decisions leading to the acquisition of neural fate involves a
switch
between these two identities in addition to the choice between
epidermis and
neural plate, as suggested by the Spemann/Mangold transplantation
experiment
and its equivalent in other species. One possibility, therefore, is
that two
separate decisions lead to the establishment of the neural plate: one
(a
decision between mesendoderm and neural fates) at the medial edge of
the neural
plate, involving Churchill and Sip1 medially, which prevent further
gastrular
ingression, and the other (neural versus epidermis) at its
lateral/anterior
edges, which could involve inhibition of BMP signaling.

A view from the
border

The same screen that
led to the
identification of Churchill also identified another early
response to an
organizer graft: ERNI (Early Response to Neural Induction) (Streit et al., 2000).
Like Churchill, this is expressed in the
prospective neural plate but its expression begins much earlier, before
gastrulation. At the end of gastrulation ERNI is down-regulated
starting
from the center of the neural domain until it is expressed only at the
neural-epidermis border, and then it disappears from this domain after
stage 7.
Also like Churchill, ERNI is induced by a graft of the
node and
by FGF, but this induction is much more rapid (just 1 hour). Analysis
of the
expression of different FGFs and comparison with these early
“pre-neural”
markers suggested that ERNI and Sox3 are first induced
before
gastrulation, by FGF emanating from either the hypoblast or from
prospective
organizer cells at the posterior end of the blastodisc or both (Streit et al., 2000).
Indeed, transplantation of either of these tissues
can induce ERNI and Sox3 just like a node or FGF, and
blocking
the FGF pathway abolishes their induction by any of these tissues.
These and
other findings (Streit and Stern,
1999a)
led to the view that an early response to neural
induction (and FGF signaling) is the specification of a region with
“border-like” character, which is responsive to BMP and its antagonists
(Streit et al., 1998; Streit and Stern,
1999a, b).
Subsequent events confine these properties
exclusively to the future neural/epidermal border, as the neural plate
proper
becomes insensitive to BMP during gastrulation. By stage 3+-4,
the
only region sensitive to BMP signaling is the border itself:
up-regulation of
BMP here moves the border towards the midline (but only by a modest
amount),
narrowing the neural plate, while down-regulation of BMP at the border
widens
the neural plate (but again only slightly). Sip1 was first identified
by its
interaction with phosphorylated Smad1, a target of BMP (Verschueren
et al., 1999),
raising the possiiblity that Churchill, through
Sip1, contributes to sensitizing cells to BMP antagonists after 5
hours’
exposure to FGF.

The
situation in amphibians may not be different from that in birds. Based
on at
least some fate maps from early (32-64-cell) stages in both Xenopus (Jacobson and Hirose, 1981; Moody, 1987;
Moody and Kline, 1990)
and in other amphibians (Moury and Jacobson, 1989,
1990; Saint-Jeannet and Dawid, 1994;
Delarue et al., 1997),
the most animal blastomeres (A2 and A3) will
contribute progeny to the neural crest (i.e. the border of the neural
plate).
Since most injections designed to test the ability of BMP antagonists
and other
factors to induce a neural plate are placed in the animal pole, it is
likely
that they target, at least in part, what may be the most sensitive
region: the
neural/epidermal border. It is also possible that the observations that
cutting
an animal cap activates MAPK as well as inducing expression of cement
gland
markers (the cement gland is part of the anteriormost border of the
neural
plate; see above) are causally connected. In agreement with this view,
injection of the dowstream inhibitory component of the BMP pathway, Smad6
in Xenopus at the 32-cell stage causes axis duplications and/or
expansion of the neural plate when placed into the A1-A3 animal
blastomeres,
but no ectopic neural plate when placed into the most ventral, A4
animal
blastomere (Delaune et al., 2004;
Linker et al., 2004).
Together these observations raise the possibility
that inhibition of the BMP pathway may only be effective in generating
ectopic
neural plate (expansion of the endogenous neural plate) within or close
to the
border between neural and epidermal territories, but not within a
region wholly
destined to give rise to epidermis. The results also point to the
border of the
neural plate as a special region, distinct from both neural plate and
epidermis.

The timing of
neural induction

Organizer grafts are
technically
easiest after the start of gastrulation, when the blastopore, primitive
streak
or shield can be identified morphologically. Experiments in which the
stage of
the host and donor embryos were varied in different vertebrate classes
established that neural induction is likely to end by the end of the
gastrula
stage. For example, in the chick, a Hensen’s node taken from an embryo
up to
stage 4 can induce a complete nervous system but older donors gradually
lose
their inducing ability, while hosts rapidly lose their competence
between
stages 4 and 4+ (Damas,
1947; Gallera and Ivanov, 1964; Gallera, 1971; Dias and Schoenwolf,
1990;
Storey et al., 1992; Streit et al., 1997).
Experiments such as these have suggested that neural
induction by the organizer is likely to end by the end of gastrulation,
but do
not give insight into when the process starts. As mentioned above, at
least
some of the early signals may be present and active before the start of
gastrulation (Streit et al., 2000;
Wilson et al., 2000).
At this time, a number of “pre-neural” genes are
expressed in the epiblast (including ERNI, Sox3 and Otx2),
in a fairly broad domain which includes but is not restricted to the
future
neural plate. Some cells expressing all three markers are destined to
contribute to mesendoderm as well as neural/epidermis border and some
epidermis. Further signals and other refining mechanisms are required
downstream of this initial “pre-neural” specification to commit cells
to a
neural fate.

In
the chick, grafts of the hypoblast (which expresses both FGF8
and the
Wnt antagonists Dkk-1, crescent and Cerberus)
can induce
all three “pre-neural” genes, but only transiently (Foley
et al., 2000; Streit et al., 2000).
It has been suggested (Stern, 2001)
that signals from the node or from its derivatives
like the head process/notochord and/or the prechordal mesendoderm may
be
required to stabilize this early expression and to drive cells to Sox2
expression and commitment to a neural plate fate. The hypoblast is
equivalent
(in terms of fate as well as expression of various markers) to the
Anterior
Visceral Endoderm (AVE) of the mouse (see Avian
gastrulation), which has been shown to be required for normal
development of the mouse
forebrain (Thomas and Beddington,
1996; Beddington and Robertson, 1998, 1999).
However, it is important to point out that neither
the chick hypoblast nor the mouse AVE are equivalent to Spemann’s
organizer in
that neither can induce the formation of a neural plate/axis when
grafted to an
ectopic site. In the mouse, it was shown that forebrain induction
requires a
combination of the AVE, the “early gastrula organizer” (EGO – which may
contain
some of the precursors of the later node) as well as the appropriate
responding
part of the epiblast (prospective forebrain) (Tam
and Steiner, 1999).

The
findings
that grafts of a mouse node to a lateral site induce a nervous system
lacking
the most rostral structures (Beddington,
1994)
and that homozygous HNF3β mutants, which lack
a node (Klingensmith et al., 1999),
still have a fairly acceptable neural tube, have
been used by some to argue that the node is not essential for neural
induction.
Indeed, it is clear that the most rostral portions of the neural plate
(prospective forebrain) are never adjacent to the mature node in either
mouse
or chick embryos. However, the former finding can be explained because
mouse
embryos are very small and it is virtually impossible to find a site to
graft
the node allowing a complete axis to form without fusing with the host,
and the
latter can be explained by the possibility that although no
morphological node
or its derivatives form, some of its properties are also expressed by
other
tissues. Furthermore, by the time a “node” can be defined
morphologically in
the mouse, the embryos are starting to produce a head process; chick
nodes at
the equivalent stage (4+/5) have already lost their ability to induce
the most
rostral parts of the CNS (Dias and
Schoenwolf, 1990; Storey et al., 1992).
Taking this evidence together, the most parsimonious
view is therefore that during normal development, signals from the AVE
and/or
other areas initiate some of the earliest events of neural induction,
but are
not sufficient. As development proceeds to gastrulation, “maintenance”
signals
as well as regional identity are imparted by the node and/or its
derivatives
(prechordal and chordal tissues) (Stern,
2001).
However, the node itself (provided that it is taken
from an embryo at the full primitive streak stage but before any
prechordal/head process cells emerge) contains sufficient signals to
induce a
complete axis when grafted far enough from the host neural plate. In
the chick
this can be done in the inner third of the area opaca, which only has
extraembryonic fate (Gallera, 1971;
Dias and Schoenwolf, 1990; Storey et al., 1992; Streit et al., 1998);
in the mouse (and perhaps also in Xenopus), it is
impossible to find a site far enough from the host neural plate to
avoid
recruitment of host neural plate cells.

Conclusions

Although very
substantial
progress has been made in understanding neural induction since the
pioneering
experiments of the 1920s, there are still substantial gaps in our
knowledge of
both the cellular and molecular aspects of this important process. It
is now
becoming more likely than rather than a single inducing factor, a whole
cascade
of molecular events and converging pathways are required to specify the
neural
plate, and that different mechanisms may contribute to define this
territory in
different embryonic locations at different times. Our view is that a
full
understanding will only come when the embryological/cellular processes
can be
fully correlated with their molecular basis – much the same as what
Wolpert may
have had in mind when criticizing excessive emphasis on signaling
events
(“inductions”) at the expense of understanding the resulting patterns: “…
induction and its related concepts, which have so dominated
embryological
thinking, have completely obscured the problems of pattern formation by
emphasizing the information coming from some other tissue rather than
the
response in the tissue which gives rise to the pattern … {a} failure of
inductive theory to consider the problem of spatial organization”
(Wolpert,
1970; quoted from Horder, 2001).

Fig. 1:
Diagram of the
“organizer graft” experiment of Spemann & Mangold (1924). The
dorsal lip
(red) of the blastopore (thick black line) of a donor newt at
mid-gastrula
stage is transplanted to the opposite (ventral) side of a host.

Fig. 2:
Diagrams bu Hilde
Mangold, illustrating the results of her organizer graft experiments
(Fig. 1).
The upper four figures are Indian ink drawings prepared as for
publication,
showing her embryos (Um25b, Um27a and two views of Um16) in whole
mount. The
lower part of the figure shows sketches from the sections of her most
famous
grafted embryo (Um132) where the donor tissue is colored red. Note that
the
mesoderm including somite and notochord is derived from the donor,
while the
adjacent nervous system is not. Access to the notebook and permission
to
reproduce them by courtesy of Jenny Narraway and the Embryological
Collection
of the Hubrecht Laboratory, Utrecht.

Fig. 3: The
“default
model” in Xenopus. On the left is a rough fate map of a blastula-stage
embryo
(organizer in red, ventral mesoderm in pink, neural in blue, epidermis
yellow,
yolky endoderm green). The inhibitory arrows represent BMP antagonist
activity
emanating from the organizer. On the right is a “genetic” diagram of
the
inductive interactions proposed by the model: ectoderm cells
(represented by
the grey boxes) have an autonomous tendency to differentiate into
neural
tissue, but are prevented from doing this and directed instead to
epidermis by
BMP4, which is expressed ubiquitously. Near the organizer, BMP
antagonists
block BMP4 signaling allowing neighboring ectoderm cells to develop
according
to their “default” neural fate.

Fig. 4:
Summary of the
main experiments supporting the “default model” (Fig. 3) as done in
Xenopus.
The leftmost two columns illustrate the results of cell dissociation
experiments, the next two show the effects of incubating animal caps
with BMP
antagonists, and the last two columns summarize the most common type of
“animal
cap” experiment. The lower box shows the usual results of these
experiments
where + implies expression of markers for the tissue shown, - means no
expression, and up- or down-arrows represent up- or down-regulation
respectively.

Fig. 5:
Organizer graft
experiment in the chick, also demonstrating the changes in inducing
ability of
the organizer with increasing age of the donor. The middle diagram
shows a host
chick embryo, at stage 4. This embryo simultaneously receives a graft
of a
quail stage 4 node on its left and a quail stage 6 node on its right.
The lower
panel shows the result of this experiment, after in situ hybridization
(purple)
for the hindbrain marker Krox-20 (expressed in rhombomeres 3
and 5,
arrows) and staining with an anti-quail antibody (brown). The young
graft has
induced a complete axis including the entire head, while the older
graft on the
right has generated a short axis, mostly derived from the graft itself
and
lacking rostral structures including the Krox-20-expressing
region.
Experiment performed by Kate Storey (Storey et al. 1992).

Fig. 6:
Results of chick
misexpression experiments. The upper diagram shows the two main types
of
misexpression experiments usually done in whole chick embryos: a graft
of
cultured COS cells that had been transfected with an expression plasmid
encoding a secreted factor (left), and in vivo electroporation of an
expression
plasmid encoding the desired protein (which may be a transcription
factor,
secreted protein or any other construct) directly into a test region of
the
epiblast. The lower table summarizes the results of the main
experiments done
in whole chick embryos. + indicates induction, - no induction, n.e. no
effect.
In the first three examples (node, hypoblast, FGF8) the time (hours) of
exposure required to obtain induction of the marker is shown.

Brachyury (Bra) is a
marker for
mesoderm. Sox2 is the “definitive” neural plate marker and the
remaining
markers (ERNI, Otx2, Sox3, ChCh) are expressed in the early epiblast
including
the prospective neural territory but do not indicate commitment of the
cells to
a neural fate. “αWnt” is a mixture of three different Wnt antagonists
(crescent, NFz8 and Dkk1) and a multifunctional antagonist of Wnt, BMP
and
Nodal (Cerberus). Note that no combination of factors can mimic the
induction
of Sox2 by the node.

Fig.
7: Model
summarizing the regulation and functions of ChCh during early
development. A-D:
the embryologist’s view; E: the geneticist’s view. In A-D,
embryos are
shown at four stages, with their germ layers exploded. A. At
stages
XI-XII, the hypoblast (brown) emits FGF8 which induces the early
pre-neural
genes ERNI and Sox3 (orange) in the overlying epiblast
(yellow),
but the cells in this domain are still uncommitted. At this stage Nodal
is expressed in the posterior (right) epiblast but is inhibited by
Cerberus
secreted by the hypoblast. B. At stages XIII-2, the hypoblast
is
displaced from the posterior part of the embryo by the endoblast
(white) which
allows Nodal signaling, in synergy with FGF, to induce Brachyury
and Tbx6L
and ingression (red arrows) to form the primitive streak (red). C.
At
stages 3+-4, continued FGF signaling now induces Churchill
in
a domain of the epiblast (turquoise). The border of the epiblast
territory
destined to ingress to form mesoderm is shown with a dashed black line.
D.
At the end of stage 4, Churchill induces Sip1, which
blocks Brachyury,
Tbx6L and further ingression of epiblast into the streak. The
epiblast
remaining outside the streak (blue) is now sensitized to neural
inducing
signals emanating from the node (blue arrows). E. The same
model shown
as a genetic cascade. Interactions described in this paper are shown as
black
lines, those from the literature are faint. The time axis runs
vertically,
wherein the color gradients indicate progressive commitment to
epidermis
(yellow), neural (blue) and mesoderm (red). BMP/Smad/Sip1 interactions
regulate
the epidermis-neural plate border, while ChCh/Sip1/FGF/Bra/Tbx6
regulate the
mesoderm-neural decision. Reproduced from Sheng et al. (2003) with
permission
of Cell Press.